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DOI:10.1016/j.yexcr.2016.06.008
Document VersionPeer reviewed version
Link to publication record in King's Research Portal
Citation for published version (APA):Zhang, Q., Minaisah, R-M., Ferraro, E., Li, C., Porter, L. J., Zhou, C., ... Warren, D. T. (2016). N-terminalnesprin-2 variants regulate -catenin signalling. Experimental Cell Research. DOI: 10.1016/j.yexcr.2016.06.008
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Download date: 21. Jun. 2018
Author’s Accepted Manuscript
N-terminal nesprin-2 variants regulate β-cateninsignalling
Qiuping Zhang, Rose-Marie Minaisah, ElisaFerraro, Chen Li, Lauren J. Porter, Can Zhou, FangGao, Junyi Zhang, Dipen Rajgor, Flavia Autore,Catherine M. Shanahan, Derek T. Warren
PII: S0014-4827(16)30158-6DOI: http://dx.doi.org/10.1016/j.yexcr.2016.06.008Reference: YEXCR10262
To appear in: Experimental Cell Research
Received date: 19 October 2015Revised date: 13 June 2016Accepted date: 14 June 2016
Cite this article as: Qiuping Zhang, Rose-Marie Minaisah, Elisa Ferraro, Chen Li,Lauren J. Porter, Can Zhou, Fang Gao, Junyi Zhang, Dipen Rajgor, FlaviaAutore, Catherine M. Shanahan and Derek T. Warren, N-terminal nesprin-2variants regulate β-catenin signalling, Experimental Cell Research,http://dx.doi.org/10.1016/j.yexcr.2016.06.008
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www.elsevier.com/locate/yexcr
1
N-terminal nesprin-2 variants regulate β-catenin signalling.
Qiuping Zhang, Rose-Marie Minaisah, Elisa Ferraro, Chen Li, Lauren J Porter, Can
Zhou, Fang Gao, Junyi Zhang, Dipen Rajgor, Flavia Autore, Catherine M Shanahan and
Derek T Warren
British Heart Foundation Centre of Research Excellence, Cardiovascular Division,
King’s College London, UK.SE5 9NU.
+Corresponding author: Dr. Derek Warren
Kings College London
Division of Cardiovascular Medicine,
James Black Centre
125 Coldharbour Lane
London SE5 9NU
UK
Tel: +44 020 7848 5222
Fax: +44 020 7848 5193
Email: [email protected]
Key Words: Nesprin-2, β-catenin, cell-cell junctions, scaffold protein
Word count: 4,882
2
Abstract
The spatial compartmentalisation of biochemical signalling pathways is essential
for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as
signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-
2 N-terminal variants. We show that these nesprin-2 variants display cell specific
distribution and reside in both the cytoplasm and nucleus. Immunofluorescence
microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at
cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2
and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin
localisation at the NE remained unaltered, furthermore, an N-terminal fragment of
nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin.
Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss
of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and
augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack
nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin
signalling independently of the NE. Together, these data identify N-terminal nesprin-2
variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell
contacts to inhibit β-catenin transcriptional activity.
Abbreviations
NE, Nuclear Envelope; ONM, Outer nuclear membrane; INM, Inner nuclear membrane;
F-actin, filamentous actin; EDMD, Emery–Dreifuss muscular dystrophy; CHD, Calponin
homology domain; SR, Spectrin repeat; LINC, Linker of nucleoskeleton and
cytoskeleton; WB, Western blot; IF, Immunofluorescence microscopy; IP,
Immunoprecipitation; ESC, Embryonic stem cells; VSMC, human vascular smooth
muscle cell; HDF, human dermal fibroblast cell; HUVEC, human umbilical vein
endothelial cells.
3
1. Introduction
Nesprins are a family of spectrin repeat containing proteins that are encoded by
four genes (SYNE1-4) [1-4]. Nesprins-1 and -2 are highly complex and multiple variants
arise due to alternative initiation and termination of the genes [5]. The giant nesprin-1
and -2 variants consist of an N-terminal paired Calponin Homology domain (CHD) that
has been shown to bind filamentous actin (F-actin), a central rod region composed of
numerous spectrin repeats and a C-terminal Klarsicht, ANC-1, SYNE Homology (KASH)
domain that is required for the nuclear envelope (NE) localisation of these proteins [4, 6,
7]. To date, the best studied function of these proteins is at the NE, where smaller
variants function to organise the inner nuclear membrane (INM) via interactions with
lamins A/C and emerin [6, 8], whereas the nesprin giant variants reside on the outer
nuclear membrane (ONM) and are components of the LInker of Nucleoskeleton to
Cytoskeleton (LINC) complex. The LINC complex physically couples the ONM to the
INM via interactions between the KASH domain of nesprins and the SUN domain of
SUN1/2 in the perinuclear space [9, 10]. SUN1/2 span the INM and interact with lamins
A/C [11, 12], thus forming a continuous biophysical network between the cytoskeleton
and nucleoskeleton [10-12]. In addition to the giant nesprin-1 and -2 isoforms, nesprin
variants that lack the KASH domain have been shown to localise to the cytoplasm and
nucleoplasm [5, 13-15]. Although the functions of these KASH-less variants remain to
be fully defined, they show tissue and cell specific expression patterns, suggesting
nesprins are tailored for specific cellular functions.
Nesprins are comprised of multiple spectrin repeats that are proposed to mediate
protein-protein interactions, however, our knowledge of nesprin binding partners
remains limited [16]. At the INM, nesprin variants interact with lamins A/C, SUN1/2 and
emerin [6, 12]. Mutations in these nesprin variants result in emerin mislocalisation,
nuclear morphology defects and are associated with Emery–Dreifuss muscular
dystrophy (EDMD), suggesting that nesprins perform a scaffolding role at the NE [1].
KASH-less variants also perform a scaffolding role in the nuclear interior and we have
previously identified nesprin-2 as a nuclear ERK scaffold that tethers ERK1/2 at
promyelocytic leukaemia nuclear bodies to regulate proliferation [14]. Importantly,
4
several cytoplasmic binding partners have also been identified for nesprin-1 and -2
including the RNA binding proteins Dcp1a, Rck and Ago2, and meckelin, respectively
[13, 17]. Moreover, nesprin-1 and -2 KASH-less variants localise to focal adhesions and
actin/microtubule filaments, suggesting that the cytoplasmic KASH-less variants may
perform a similar scaffolding role [5, 13]. Nesprin-2 has also been implicated in the WNT
pathway that transfers signals from the plasma membrane to the nucleus via nuclear
translocation of the transcription factor β-catenin [18-21]. Both α- and β-catenin interact
with spectrin repeats (SRs) toward the C-terminus of nesprin-2 giant to attenuate β-
catenin signalling [22]. In addition to this direct interaction, nesprin-2 may also indirectly
associate with β-catenin at the INM, where the nesprin-2 binding protein emerin
interacts with β-catenin to facilitate its nuclear export [23].
In this study we investigate the role of recently identified N-terminal nesprin-2
variants that retain the CHD but lack the KASH domain. We show that these variants
are novel components of cell-cell junctions, where they colocalise and interact with β-
catenin. Importantly, these nesprin-2 variants anchor β-catenin to cell-cell junctions to
negatively regulate β-catenin mediated transcriptional activity.
2. Materials and Methods
2.1. Cell culture
Human bone osteosarcoma epithelial (U2OS), human umbilical vein endothelial
cells (HUVEC), mouse C2C12 myoblast, human dermal fibroblast and human vascular
smooth muscle cells were cultured as described previously [24, 25]. The following
nesprin-2 siRNA oligomers targeting the N-terminus of the giant variant were used in
this study: siN2CH2 (5’ AGGAAGACACCCAGAAGUU 3’), siN2CH3 (5’
CUUCAGAAUUGCAGAACAAUU 3’), siN2CH5 (5’ GCCUUCACGUGCUGGAUAAUU
3’), p220CHNesp2 3’UTR1 (5’ GAGAAUAGUCUGUGGAGAAUU 3’), p220CHNesp2 3’UTR2
(5' GGAACGUAGUGGAGGAUAUU 3'), p380CHNesp2 3’UTR1 (5'
AUCGAAAGCCAGAGAGUAAUU 3') and p380CHNesp2 3’UTR2 (5'
AGUCAGAGGUCAACAACAAUU 3') (Dharmacon). C-terminal nesprin-2 siRNA
5
designed to a region close to the KASH domain (siN2KASH) have been described
previously [14]. Emerin smart pool siRNA oligomers from Dharmacon were used in this
study. Transfection of siRNA was performed using HiPerfect (Qiagen), as per
manufacturer’s instructions. DNA transfections of were performed with Fugene
(Promega) as per manufacturer’s instructions.
2.1. PCR and 3'UTR amplification
PCR for N-terminal nesprin-2 3'UTRs were performed using 3’UTR specific
primer sets as described previously [5].
2.3. Nesprin constructs
The following N-terminal nesprin-2 fragments were cloned into pEGFP-C1 vector
(Clontech): ABDN2 (amino acids 1-531). The CHDN2 (amino acids 1-278) fragment
was cloned into the pCMV-Tag vector (Agilent Technologies). The SR 1-3 region (amino
acids 279-531) was cloned into the pGEX4T-1 (Amersham) and pCMV-Tag (Agilent
Technologies) vectors.
2.4. Calcium switch assay
Cells were grown to 80-100% confluency and serum starved overnight. Next day,
cells were incubated with 4mM EGTA in calcium free media for 1 hour to promote
cadherin mediated cell-cell junction disassembly. Junction re-assembly was promoted
by incubating cells in media containing 1.8mM calcium for 1 hour. Cells were fixed and
processed for immunofluorescence microscopy.
2.5. Western blot analysis, antibodies and Immunofluorescence microscopy
Cell lysates were run on 5% or 8% polyacrylamide gels and subjected to Western
blotting as described previously [6]. Antibodies used for Western blot, confocal
immunofluorescence microscopy (IF) and immunoprecipitation were; GFP (ab290),
GFP-Sepharose (ab69314) (Abcam), Vinculin (Sigma), Emerin (VP-E602) (Vector
Labs), lamin A/C (sc-6215) (Santa Cruz), total β-catenin, active β-catenin clone 8E7
(05-665) (Millipore), nesprin-2 CH3 and nesprin-2 N3 (Immune Systems). N2CH3
6
peptide blocking experiments were performed as described previously using the peptide
KRDLDELKDHLQL (Immune Systems) [6]. Filamentous actin was observed by IF using
Rhodamine phalloidin (Invitrogen). Secondary antibodies for WB were horseradish
peroxidase-conjugated anti mouse (NA931) or anti rabbit (NA94V) antibodies from GE
Healthcare. ECL chemiluminescent kit (RPN2132, GE Healthcare) was used for
detection according to manufacturer’s instructions. Invitrogen anti-mouse Alexa fluor
568 (A11031) and anti-rabbit Alexa fluor 488 (A11034) were used as IF secondary
antibodies. For IF cells were cultured on cover slips, fixed in 4% paraformaldehyde
(Sigma), permeabilised in 0.5% NP-40 (Sigma) and processed as described previously
[6]. All images were captured at 63 x magnifications using a Leica SP5 laser scanning
confocal microscope.
2.6. Immunoprecipitation, GST pull-downs and subcellular fractionations
Subcellular fractionations were performed as described previously [14]. GST
expression, purification and pull-down assays were performed as described previously
[14]. For immunoprecipitation (IP), U2OS cells were transfected with either GFP or
GFP-ABDN2 and incubated overnight. Cells were processed for IP as described
previously [14]. GFP was immunoprecipitated by incubating with anti-GFP coated
Sepharose beads for 2 hours at 4ºC. Beads were washed three times in IP buffer before
bound proteins were eluted in sample buffer, as described previously [14]. Coomassie
staining was performed using the Bio-SafeTM Coomassie stain (BIORAD) as per
manufacturer’s instructions.
2.5. Luciferase assays
U2OS cells were seeded onto a 6 well plate at a density of 2.5x105 cells per well.
Next day cells were transfected with mixtures of 1µg TOP-FLASH or FOP-FLASH,
0.1µg TK Renilla and 1µg of GFP, GFP-ABDN2, FLAG or FLAG-SR 1-3 using Fugene
(Promega). Cells were incubated overnight. For analysis of siRNA on transcriptional
activity the TOP-FLASH or FOP-FLASH and TK Renilla mix was added directly to
siRNA transfection mixture containing HiPerfect (Qiagen). Cells were incubated for 48
hours. Luciferase and Renilla activities were assayed using the Dual-Glo® Luciferase
7
assay system (Promega) as per manufacturer's instructions. Control Luciferase
activities were assigned a value of 1.
2.6. Statistical analysis
Results are presented as mean +/- SEM. For comparison of siRNA knockdown groups
paired Student’s t-tests or one way ANOVA with Bonferroni’s post-test were performed.
3. Results
3.1 Cell specific distribution of Nesprin-2 variants.
Recently, 3’UTRs encoding KASH-less N-terminal nesprin-2 variants
(p220CHNesp2 and p380CHNesp2) were identified by EST data base searches (Figure 1A).
These 3’UTRs display tissue specific expression patterns [5]. To describe the cell
specificity of these 3'UTRs, we performed PCR analysis and we show that p220CHNesp2
is abundant in human bone osteosarcoma epithelial (U2OS) and vascular smooth
muscle cells (VSMC) but absent in human dermal fibroblast (HDF) and mouse C2C12
myoblast cells. The p380CHNesp2 variant was abundant in U2OS, HDF and myoblast
cells, but lacking in human umbilical vein endothelial cells (HUVEC) and VSMCs (Figure
1B). Western blot analysis (WB) was performed on whole cell lysates using an antibody
raised to the N-terminus of the nesprin-2 giant (N2CH3) (Figure 1C). To confirm the
specificity of the N2CH3 antibody we performed peptide blocking experiments and show
that the activity of the antibody is efficiently blocked by the target sequence on WB
(Supplementary Figure 1A). In agreement with the PCR data, we show that U2OS cells
possess both the p220CHNesp2 and p380CHNesp2 variants whereas VSMCs and HDFs
possess either the p220CHNesp2 or p380CHNesp2 variant, respectively (Figure 1D).
Importantly, using the N2CH3 antibody and a C-terminal nesprin-2 antibody (N2N3) we
show that the nesprin-2 giant is highly abundant in VSMCs but was not detectable in
U2OS and HDF cells tested (Figures 1C and D). As previous studies have shown that
the nesprin-2 giant is present in HDF cells at low levels, we performed subcellular
fractionation experiments to concentrate the nuclear proteins [26]. WB revealed that
8
nesprin-2 giant was weakly present in HDF nuclear fractions. Importantly, nesprin-2
giant was not detected in U2OS nuclear fractions, further confirming that U2OS cells
lack nesprin-2 giant (Supplementary Figure 1B).
Subcellular fractionation of U2OS cells demonstrated that p220CHNesp2 and
p380CHNesp2 reside in both the cytoplasmic and nuclear fractions (Figures 2A). In
addition, smaller unknown nesprin-2 bands were observed in the cytoplasmic (55kDa)
and nuclear (60 and 70kDa) fractions (Figure 2A). The p220CHNesp2 variant was also
detected in both nuclear and cytoplasmic fractions in VSMCs (Figure 2B), however, the
p380CHNesp2 variant was nuclear in HDF cells (Figures 2C). In all cell types tested,
unknown variants were detected, suggesting that our knowledge of nesprin-2 variants
remains incomplete (Figures 2A-C).
3.2. Nesprin-2 variants localise to cell-cell junctions and interact with β-catenin.
Next, we employed confocal fluorescence microscopy (IF) to investigate the
cellular localisations of these variants. IF demonstrated that the nesprin-2 antibody
raised to the N-terminus of nesprin-2 giant (N2CH3) diffusely stained within the nucleus
and at the sites of cell-cell contact at the cell periphery, where nesprin-2 colocalised
with active β-catenin in U2OS cells (Figures 3A and B). In contrast, no colocalisation
with β-catenin was observed in HDF cells (Supplementary Figure 2). To investigate the
significance of nesprin-2 localisation at cell-cell contacts further, U2OS cells were grown
in high or low calcium conditions to promote or inhibit cadherin mediated cell junction
formation respectively. IF revealed that, U2OS cells in the presence of high calcium,
displayed colocalisation of nesprin-2 and active β-catenin at cell-cell junctions, however,
localisation of both nesprin-2 and β-catenin is rapidly lost from the plasma membrane
when cells were switched to low calcium conditions to promote cadherin disassembly
(Figure 3B). Localisation of nesprin-2 and β-catenin at cell-cell contacts was rescued by
replenishing calcium levels (Figure 3B).
To further interrogate the localisation of nesprin-2 variants that retain the CHD
but lack the KASH domain, we employed an overexpression strategy using an N-
terminal nesprin-2 construct that possessed the CHD and the antibody binding region
(amino acids 1-531) (Figure 3A). IF demonstrated that the N-terminal fragment (GFP-
9
ABDN2) colocalised efficiently with active β-catenin at cell-cell junctions in U2OS
(Figure 3C, left panel) and HDFs (Figure 3C, right panel). Importantly a similar fragment
of nesprin-1 failed to localise to cell-cell junctions and was predominantly nuclear,
suggesting that cell-cell junction localisation is specific for nesprin-2 (Supplementary
Figure 3). To further define the requirements for cell-cell junction localisation, we next
expressed the CHD region (amino acids 1-279) of nesprin-2. IF revealed that the CHD
localised to cell-cell junctions, although some stress fibre staining was also observed
(Figure 3C).
Next, we investigated whether nesprin-2 interacted with β-catenin by performing
immunoprecipitation experiments. WB revealed that β-catenin was precipitated by the
GFP-ABDN2 fragment but not GFP-alone (Figure 4A and B). Conversely, the GFP-
ABDN2 fragment was efficiently precipitated by β-catenin IP, confirming the nesprin-2 is
a novel β-catenin interacting protein (Figure 4C). Next, we mapped the β-catenin
binding site by fusing the SR region of the ABDN2 construct (SR 1-3 containing amino
acids 278-531) to GST (Figure 4A). GST pull down assays confirmed that β-catenin was
precipitated by GST-SR 1-3, but not GST alone (Figure 4D), confirming that this
spectrin repeat region interacts with β-catenin.
3.3. Nesprin-2 disruption induces cell-cell junction disassembly and augments β-catenin
transcriptional activity.
We next investigated the impact of nesprin-2 disruption on β-catenin localisation
by utilising a siRNA mediated knockdown strategy that targeted nesprin-2 variants
containing the CHDs. U2OS cells were transfected with either control or nesprin-2
specific siRNA that targeted the N-terminus of p220CHNeps2 and p380CHNesp2 (Figure
5A). WB analysis confirmed knockdown of p220CHNesp2 using 3 independent nesprin-2
specific siRNAs (Figure 5B and C). Levels of the p380CHNesp2 variant remained
unaltered by our siRNA strategy (Figure 5B and D), suggesting that p380CHNesp2 is
more stable than p220CHNesp2. WB also revealed that protein levels of C-terminal
variants that lack the siRNA target sequence remain unaltered (Figure 5B). Importantly,
levels of active β-catenin and total β-catenin remained unaltered by our nesprin-2
depletion strategy (Figure 5B). To specifically target the p220CHNesp2 and p380CHNesp2
10
variants we designed siRNAs targeting the unique 3’UTRs, however, WB revealed that
this strategy was unsuccessful and failed to deplete the p220CHNesp2 and p380CHNesp2
variants (Supplementary Figure 4).
IF was performed to observe whether nesprin-2 depletion altered β-catenin
organisation in U2OS cells and revealed that nesprin-2 depleted cells displayed
reduced staining of active β-catenin at cell-cell junctions compared to control cells
(Figure 6A, B and Supplementary Figure 5A), suggesting that the p220CHNesp2 variant
tethers active β-catenin to the sites of cell-cell contact. Importantly, subcellular
fractionation revealed that nesprin-2 depleted cells displayed increased levels of nuclear
active β-catenin (Figure 6C) and TOP-FLASH/FOP-FLASH luciferase assays confirmed
that nesprin-2 depleted cells possessed augmented β-catenin transcriptional activity
compared to control cells (Figure 6D). In contrast, siRNAs targeting the C-terminus of
nesprin-2 giant that the p380CHNesp2 and p220CHNesp2 variants lack, failed to alter
luciferase activity (Figures 6A and D), supporting the notion that N-terminal variants are
responsible for localising β-catenin to cell-cell junctions. Next, we assessed the impact
of overexpression of the β-catenin binding fragments of the N-terminal nesprin-2
variants on β-catenin signalling. However, TOP-FLASH/FOP-FLASH luciferase assays
revealed that the β-catenin binding fragments had no impact on β-catenin transcriptional
activity (Supplementary Figure 5B).
3.4. β-catenin localisation at cell-cell junctions is independent of emerin.
Previous studies have shown that nesprin disruption triggers nuclear morphology
defects, so we next performed IF to observe if our siRNA strategy altered nuclear
morphology. Analysis revealed that control cells contained spherical nuclei, however,
nesprin-2 depleted nuclei possessed a more convoluted morphology (Figures 7A and
B). Next, we performed IF to observe the localisation of the nesprin-2 interacting protein
emerin and show that nesprin-2 depleted cells display normal NE emerin staining
(Figures 7C). As emerin has previously been implicated in β-catenin signalling, we
further investigated whether changes in β-catenin signalling were due to impaired
emerin function by performing emerin knockdown experiments. WB confirmed efficient
11
emerin depletion in U2OS cells (Figure 8A), however, β-catenin organisation and
transcriptional activity remained unaltered in emerin depleted cells (Figure 8B and C).
4. Discussion
Nesprins have emerged as signalling scaffold proteins that localise to multiple
subcellular compartments, including the NE, cytoplasm and nucleoplasm [5, 6]. In this
current study, we show that nesprin-2 N-terminal variants colocalise with β-catenin at
cell-cell junctions. We show that a fragment containing the CHD and SR1-3 region
(ABDN2) was sufficient for both β-catenin binding and cell-cell junction localisation.
Further mapping identified the N-terminal SRs 1-3 of nesprin-2 as a novel β-catenin
binding region, although we did not rule out the possibility that the CHD and β-catenin
also interact. We propose that the N-terminal nesprin-2 KASH-less variants tether β-
catenin at cell-cell junctions and inhibit β-catenin transcriptional activity. In support of
this notion, nesprin-2 depleted U2OS cells displayed loss of β-catenin from cell-cell
contacts, accumulation of active β-catenin in the nucleus and augmented β-catenin
transcriptional activity. Importantly, levels of active β-catenin remained unchanged in
nesprin-2 depleted U2OS cells, suggesting that nesprin-2 depletion triggers
redistribution of active β-catenin from cell-cell contacts to the nucleus. Our
overexpression experiments show that N-terminal CHD containing nesprin-2 fragments
localise to cell-cell contacts, colocalise with active β-catenin but did not alter β-catenin
transcriptional activity. N-terminal nesprin-2 fragments that lack the CHD but retain the
β-catenin binding site also failed to alter β-catenin transcriptional activity. This suggests
that the association between active β-catenin and the N-terminal nesprin-2 variants at
cell-cell contacts is stable, further experimentation is required to elucidate the
functions/dynamics of these N-terminal nesprin-2 variants at cell-cell contacts.
We also demonstrate that the p220CHNesp2 and p380CHNesp2 variants, that
contain the β-catenin binding domain, reside in both the nucleus and cytoplasm, raising
the intriguing possibility that these KASH-less variants may shuttle between these
compartments. However, further investigation is now required to clarify whether KASH-
12
less nesprin-2 variants associate with and organise other components of the β-catenin
pathway, as well as to identify the exact nesprin-2 variant. Our knockdown strategy
efficiently depleted p220CHNesp2 but not p380CHNesp2 and presumably these two
variants display differences in protein turnover as both contain the target sequence.
This suggests that p220CHNesp2 is potentially a good candidate for future investigation
however, our siRNA strategy targeted multiple nesprin-2 variants so the possibility
remains that an unidentified variant may localise to cell-cell contacts and regulate β-
catenin signalling.
N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE
Nesprin-2 variants organise the NE and several recent studies have identified the
NE as a novel regulator of β-catenin signalling [22, 23, 30]. Firstly, β-catenin interacts
with the C-terminus of the nesprin-2 giant to positively regulate β-catenin signalling [22].
In addition, the nesprin-1/2 orthologue ANC1 regulates β-catenin signalling during
neuronal development in C. elegans [30]. Due to the sequence identity between
nesprin-2 variants, our knockdown strategy potentially targeted both KASH-less N-
terminal and the KASH-containing nesprin-2 giant variants [5]. Importantly, we show
that U2OS cells lack nesprin-2 giant and β-catenin transcriptional activity was enhanced
by N-terminal nesprin-2 depletion in U2OS cells. These data suggest that the N-terminal
nesprin-2 variants negatively regulate β-catenin transcriptional activity in U2OS cells
and highlight the complexity of nesprin-2 function in regulating β-catenin signalling.
Previous studies have also demonstrated that emerin interacts with both the C-terminal
nesprin-2 variants and β-catenin at the INM to negatively regulate β-catenin mediated
transcription [23]. KASH-containing nesprin-2 variants are essential for emerin
organisation at the NE [6], however, emerin organisation was unaltered by depletion of
N-terminal nesprin-2 variants in U2OS cells, suggesting that changes in β-catenin
signalling induced by our siRNA strategy were NE independent. Furthermore, emerin
depletion in U2OS cells failed to displace β-catenin from cell-cell junctions or alter β-
catenin transcriptional activity, further suggesting that the N-terminal nesprin-2 variants
regulate β-catenin signalling independently of the NE.
13
Despite our evidence showing that nuclear envelope function is not disrupted by
our nesprin-2 siRNA strategy, nuclear morphology was altered by our approach.
Previous studies have demonstrated that actomyosin, cell morphology and adhesion all
contribute to defining nuclear morphology and potentially, in addition to disrupting cell-
cell contacts, our nesprin-2 depletion strategy induced cytoskeletal reorganisation that
altered nuclear morphology [27, 28]. However, the potential role of the nesprin-2 N-
terminal variants in cytoskeletal organisation remains untested.
Nesprin-2 variants fine tune β-catenin signalling for cell specific functions?
Nesprin variants demonstrate complex tissue and cell-specific distributions [5,
29]. Nesprin giant variant expression is abundant in the majority of human tissues,
except cardiac and skeletal muscle, which are enriched in shorter isoforms [29]. In
addition, the nesprin-2 epsilon-1 and epsilon-2 variants are highly expressed in
embryonic stem cells (ESC) and heart respectively [29]. Here, we confirm that the
p220CHNesp2 and p380CHNesp2 variants display cell-specific expression, suggesting that
nesprin function is tailored to specific cellular functions. In support of this, up regulation
of nesprin variants and nesprin variant switching is observed in ESC, mesenchymal
stem cell and skeletal muscle differentiation [26, 30, 31]. Nesprins have emerged as
signalling scaffolds for the ERK and β-catenin pathways and these pathways exist in
multiple cell types. Furthermore, recent evidence demonstrates the importance of the
signalling scaffolding functions of the nesprin family during development, where the
nesprin-1/2 orthologue ANC1 regulates β-catenin signalling during neuronal
development in C. elegans [32]. However, we show that the nesprin-2 giant is not
detectable in U2OS cells and potentially adaptation of nesprin variant expression may
fine tune these pathways and facilitate cell-specific signalling. In support of this, we
show that U2OS cells display high levels of the p220CHNesp2, whereas fibroblasts lack
the p220CHNesp2 variant. In addition to changes in nesprin-2 variant expression, the
p380CHNesp2 variant displayed differential compartmentalisation between U2OS and
HDF cells, therefore, differential nesprin variant expression/compartmentalisation may
contribute to cell specific functions for nesprin-2 in β-catenin signalling. Further
14
investigation is now required to clarify the cell-specific functions of nesprin variants in
regulating β-catenin signalling.
Conflicts of Interest
The authors declare that no conflicts of interest exist.
Sources of funding
This work was funded by a British Heart Foundation (BHF) program grant to CMS
(program grant number RG/11/14/29056), a BHF IBSRF awarded to DTW
(FS/11/53/29020) and a BHF project grant to QPZ (PG/11/58/29004)
Figure legends
Figure 1. Cell type specific expression of N-terminal nesprin-2 variants. A) Schematic
representation of the genomic organisation of 5’ and 3’ UTRs encoding the N-terminal
variants of nesprin-2 N-terminus. B) PCR analysis of cDNA derived from U2OS, dermal
fibroblast (HDF), vascular smooth muscle (VSMC), C2C12 myoblast and human
umbilical vein endothelial (HUVEC) cells for p220CHNesp2 and p380CHNesp2 3’UTRs. C)
Schematic representation of nesprin-2 N-terminal variant structure and N-terminal
(N2CH3) and C-terminal (N2N3) nesprin-2 antibody epitope regions. D) WB of U2OS,
VSMC and HDF whole cell lysates separated on 5% polyacrylamide gels.
Figure 2. N-terminal nesprin-2 variants reside in the cytoplasm and nucleus. WB of
U2OS, VSMC and HDF of cytoplasmic (C) and nuclear (N) fractions separated on 8%
polyacrylamide gels. * mark unidentified nesprin variant bands.
15
Figure 3. N-terminal nesprin-2 variants colocalise with β-catenin at cell-cell junctions. A)
Schematic representation of nesprin-2 CH3 antibody epitope position and nesprin-2
constructs used. B) IF of nesprin-2 (CH3) (green), active β-catenin (Aβ-catenin) (red)
and DAPI (blue) localisation in U2OS cells before, during and after calcium depletion. C)
IF of GFP-ABDN2, Flag-CHDN2 (green) and active β-catenin (Aβ-catenin) (red) in
U2OS (left panel) and fibroblast cells (right panel). Scale bar = 25µm.
Figure 4. The N-terminus of nesprin-2 interacts with β-catenin. A) Schematic
representation of the nesprin-2 constructs used. B) WB of GFP/GFP-ABDN2
immunoprecipitation. C) WB of β-catenin IP. D) WB of GST-alone and GST-SR 1-3
construct pull downs. GST-loading was shown by coomassie stain.
Figure 5. Validation of nesprin-2 depletion strategy. A) Schematic representation of
nesprin-2 CH3 and N3 antibody epitopes and the region targeted by siRNA siN2CH2-5.
B) WB of N-terminal (N2CH3) and C-terminal (N2N3) variants after control and nesprin-
2 (siN2CH2/CH3/CH4) siRNA knockdown. Samples were separated on 8%
polyacrylamide gels. Graphs show relative level of C) p220CHNesp2 and D) p380CHNesp2.
Graphs represent combined data from 3 independent siRNA experiments for fold
change in densitometry ratio (*p=<0.05, **p=<0.001).
Figure 6. Nesprin-2 is required for β-catenin localisation at cell-cell junctions and
negatively regulates β-catenin transcriptional activity. A) Schematic representation of
siN2CH2/siN2CH5 and siN2KASH target regions. B) IF of active β-catenin (Aβ-catenin)
(green), F-actin (red) and DAPI (blue) in control and nesprin-2 (siN2CH2) depleted
U2OS cells. Scale bar = 25µm. C) WB of control, siN2CH2 and siN2CH5 cytoplasmic
(C) and nuclear (N) fractions. D) TOP/FOP Luciferase assay of control, siN2CH2,
siN2CH5 and siN2KASH depleted cells. Graph shows combined data from 3
independent experiments repeated in triplicate (*p=<0.05, **p=<0.001).
Figure 7. Nesprin-2 disruption alters nuclear morphology but not emerin localisation. A)
IF of nesprin-2 (green), emerin (red), and DAPI (blue) staining of control and nesprin-2
16
depleted cells. Scale bar = 10µm. B) Quantification of number of control and nesprin-2
depleted (siN2CH2 and siN2CH5) cells displaying convoluted nuclei. Graph show
combined data from 3 independent experiments counting 300 cells per group
(**p=<0.001 and ***p=<0.0001). C) IF staining of DAPI (blue), N2CH3 (green) and
emerin (red) in control and nesprin-2 depleted U2OS cells. Scale bar = 25µm.
Figure 8. Emerin disruption does not impact on β-catenin localisation. A) WB confirming
emerin knockdown. B) IF of active β-catenin (Aβ-catenin) (green), F-actin (red) and
DAPI (blue) in control and emerin depleted U2OS cells. Scale bar = 25µm. C) TOP/FOP
luciferase assay of control and emerin depleted cells. Graph shows combined data from
3 independent experiments repeated in triplicate.
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Highlights
· N-terminal nesprin-2 variants display cell specific expression patterns
· N-terminal spectrin repeats of nesprin-2 interact with β-catenin
· N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions
· Nesprin-2 variants play multiple roles in β-catenin signalling
Figure 1.
A. B.
30 20
1 10
p32CHNesp2
50
p220CHNesp2 p380CHNesp2
Exon 5’UTR 3’UTR Exon 5’UTR 3’UTR5’
U2O
S
HD
F
Myo
bla
st
HU
VE
C
VS
MC
p380CHNesp2
GAPDH
U2O
S
HD
F
VS
MC
Myo
bla
st
p220CHNesp2
GAPDH
C.
D.
N2CH3
Spectrin repeat
CH domain
KASH domain
Nesprin-2 giant
p32CHNesp2
p220CHNesp2
p380CHNesp2
N2N3
160kDa-
250kDa-
250kDa-
160kDa-
Nesprin-2 giant
p380CHNesp2
p220CHNesp2
U2OS VSMC HDF
WB: N2CH3
WB: N2N3 Nesprin-2 giant
Figure
C. B. A.
250kDa- 160kDa- 100kDa-
75kDa-
50kDa-
Vinculin
C N
p220CHNesp2
*
* *
VSMC U2OS
250kDa- 160kDa- 100kDa-
75kDa-
50kDa-
Vinculin
Lamin A/C
C N
p220CHNesp2 p380CHNesp2
* * *
250kDa- 160kDa- 100kDa-
75kDa-
50kDa-
C N
Vinculin
p380CHNesp2
*
*
HDF
Lamin A/C Lamin A/C
Figure 2.
Figure 3.
B.
A.
C.
Aβ-catenin Merged
Start
Calcium
free
Rescue
Nesprin-2
ABDN2 (amino acids 1-531)
CHDN2 (amino acids 1-278)
N2CH3
Nesprin-2
giant
GFP
FLAG
GFP GFP-ABDN2 Flag-CHDN2
Aβ-catenin
GFP only GFP-ABDN2 FLAG-CHDN2
Aβ-catenin
Merge
25µm 25µm
25µm
Figure 4.
A.
B. C.
GFP-ABDN2
GFP
GFP-ABDN2
Start
GF
P
GF
P-A
BD
N2
GF
P
GF
P-A
BD
N2
IP
WB: β-catenin
WB: GFP
WB: Nesprin-2
ABDN2 (amino acids 1-531)
SR 1-3 (amino acids 279-531)
Nesprin-2
giant
GFP
GST
GFP-ABDN2
GF
P
GF
P-A
BD
N2
GF
P
GF
P-A
BD
N2
Input
IP:
β-cat
GFP-ABDN2
GFP
WB: GFP
WB: Nesprin-2
IgG
WB: β-catenin
D.
Start GST
GST
-SR
1-3 WB:
β-catenin-A
GST-SR 1-3
GST
Coomassie
Figure 5.
A. N2N3
siN2CH2-5
N2CH3
Spectrin repeat
CH domain
KASH domain
Nesprin-2
giant
p32CHNesp2
p220CHNesp2
p380CHNesp2
C.
D.
** *
**
B.
250kDa- 160kDa-
100kDa- 75kDa-
50kDa-
250kDa- 160kDa- 100kDa-
75kDa-
50kDa-
p220CHNesp2
p380CHNesp2
WB:
N2CH3
WB:
N2N3
β-actin
A-β-catenin
Total
β-catenin
Figure 6.
A.
C.
B.
siN2KASH
siN2CH2-5
Nesprin-2
giant
p220CHNesp2
p380CHNesp2
C.
siControl siN2CH2
*
ns
**
C N C N C N
siCont CH2 CH5
A-β-catenin
Vinculin
Lamin A/C
N2CH3
p220CHNesp2
D.
Figure 7.
A. B.
C.
siN2CH2 siControl
*** **
Figure 8.
A. C.
Emerin
β-actin
B.
ns